We propose to study biotin holoenzyme synthesis in Saccharomyces cerevisiae. Our primary objective is to elucidate the recognition mechanism which allows a single holoenzyme synthetase to incorporate biotin into many different biotin-requiring enzymes. We will use two experiment approaches: 1) The primary amino acid sequence will be determined for biotin containing peptides from two biotin-requiring enzymes of Saccharomyces - pyruvate carboxylase and urea amidolyase. 2) Mutants of Saccharomyces will be selected which lack activity for all of the biotin requiring enzymes. These mutants will be analyzed to determine whether a single mutation is responsible for the observed phenotype and whether the biochemical lesion occurs in the synthesis of a single non-specific biotin-holoenzyme synthetase.